Effect of plant extracts on H₂O₂-induced inflammatory gene expression in macrophages

Figures & data

Table 1 Abbreviation, botanical name, used part, and yield of extract of each screened plant

Extract abbreviations	Botanical name	Used part	Origin	Content (HPLC method)	Extract yield (volume/volume)
					Water	Ethanol
AL	Arctium lappa	Root	Italy	40% arctigenin	100	–
CS	Camellia sinensis	Leaf	Asia	≥95% polyphenols, ≥70% catechins total (≥40% EGCG)	–	100
EA	Echinacea angustifolia	Root	Italy	≥4% echinacoside	20	80
ES	Eleutherococcus senticosus	Root	Asia	2% eleutherosides	50	50
PG	Panax ginseng	Root	People’s Republic of China	≥30% ginsenosides tot	50	50
VM	Vaccinium myrtillus	Fruit	Italy	≥25% anthocyanins	40	60

Abbreviations: EGCG, epigallocatechin gallate; HPLC, high-performance liquid chromatography.

Table 2 Primer pair sets and parameters used in the real time polymerase chain reaction analysis

Gene	GenBank accession number	Sequence (5′ → 3′)	Product length (bp)	Primer (nM)	Tan (°C)
COX2	AK134081	For: CCCACAGTCAAAGACACTCA Rev: CTCATCACCCCACTCAGGA	189	200	57
IL1β	AK225002	For: GACCTTCCAGGATGAGGACA Rev: AGCTCATATGGGTCCGACAG	183	200	58
NFE2L2	BC152371	For: GGTTATCACAGCTGCCTG Rev: GGTAGTTCTGGGACATGG	253	200	56
NFκB1	AK090099	For: CTGACCTGAGCCTTCTGGAC Rev: GCAGGCTATTGCTCATCACA	177	200	59
NFκB2	AK144994	For: TGACTGTGGAGCTGAAGTGG Rev: GGTGTGTTTCCAGCAAAGGT	153	200	58
NOS2	M87039	For: CACCTTGGAGTTCACCCAGT Rev: ACCACTCGTACTTGGGATGC	170	200	59
PPARγ	U01841	For: GAGCTGACCCAATGGTTG Rev: ACCCTTGCATCCTTCACAAG	232	200	57
TNFα	AK155964	For: CGAGTGACAAGCCTGTAGCC Rev: GTGGGTGAGGAGCACGTAGT	209	200	60
ACTB	NM_007393	For: CACCAACTGGGACGACATGGAG Rev: GGCCTGGATGGCCACGTACAT	203	200	59
GAPDH	NM_008084	For: AATGTGTCCGTCGTGGATCTGA Rev: AGTGTAGCCCAAGATGCCCTTC	117	200	60

Abbreviations: For, forward; Rev, reverse; Tan, annealing temperature; COX2, cyclooxygenase 2; IL1β, interleukin 1β; NFE2L2, nuclear factor E2-related factor 2; NFκB, nuclear factor κB; NOS2, nitric oxide synthase 2; PPARγ, proliferator-activated receptor γ; TNFα, tumor necrosis factor α; ACTB, β actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Table 3 messengerRNA expression of TNFα, IL1β, NFκB1, NFκB2, NOS2, COX2, NFE2L2, and PPARγ in RAW264.7 cells treated with 50, 100, and 200 μM of H₂O₂ for 30 and 60 minutes in comparison to control cells

Gene	Dose (μM)	30 minutes	60 minutes	Mean squared error	Time	Dose	Time × dose
		log2 (n-fold)
TNFα	50	0.88	0.98	0.225	0.012	0.009	0.207
	100	1.07	1.81
	200	1.39	2.52
IL1β	50	1.23	0.86	0.405	0.817	0.746	0.551
	100	1.21	1.36
	200	1.08	1.52
NFκB1	50	0.89	1.56	0.464	0.000	0.008	0.124
	100	1.05	3.03
	200	1.57	3.89
NFκB2	50	0.97	1.74	0.114	0.000	0.002	0.044
	100	1.17	2.33
	200	1.36	3.23
NOS2	50	0.91	1.15	0.342	0.35	0.266	0.025
	100	1.69	1.53
	200	0.43	2.3
COX2	50	0.55	1.69	0.096	0.000	0.000	0.000
	100	0.21	2.6
	200	0.79	4.05
NFE2L2	50	0.13	2.73	0.206	0.000	0.002	0.013
	100	2.03	3.16
	200	0.85	3.70
PPARγ	50	−0.56	−0.61	0.108	0.694	0.150	0.956
	100	−0.72	−0.73
	200	−0.92	−1.04

Notes: Data are presented as the log₂(n-fold) mean ± standard error (n=3). The significance of time of incubation (30 and 60 minutes) and doses, and of the interaction between time × dose (determined by the analysis of variance), are reported.

Abbreviations: COX2, cyclooxygenase 2; IL1β, interleukin 1β; NFE2L2, nuclear factor E2-related factor 2; NFκB, nuclear factor κB; NOS2, nitric oxide synthase 2; PPARγ, proliferator-activated receptor γ; TNFα, tumor necrosis factor α.

Figure 1 messengerRNA expression of TNFα, IL1β, COX2, NOS2, NFκB1, NFκB2, NFE2L2, and PPARγ in RAW264.7 cells treated with 10 μg/ml of each extract for 24 hours after a pre-stimulation using 200 μM H₂O₂ for 1 hour relative to H₂O₂-treated cells.

Notes: (A) TNFα; (B) IL1β; (C) COX2; (D) NOS2; (E) NFκB1; (F) NFκB2; (G) NFE2L2; (H) PPARγ. To compare the bioactivities between six plant extracts for each gene, the general linear model was used with fixed effect for extracts. Bars represent the log₂(n-fold) mean ± standard error (n=4). The effect of plant extracts for each gene are indicated with different capital letters denoting significant differences at P<0.001.

Abbreviations: AL, Arctium lappa; CS, Camellia sinensis; EA, Echinacea angustifolia; ES, Eleutherococcus senticosus; PG, Panax ginseng; VM, Vaccinium myrtillus; COX2, cyclooxygenase 2; IL1β, interleukin 1β ; NFE2L2, nuclear factor E2-related factor 2; NFκB, nuclear factor κB; NOS2, nitric oxide synthase 2; PPARγ, proliferator-activated receptor γ; TNFα, tumor necrosis factor α.

Table 4 Cell viability of RAW264.7 cells treated with 50, 100, and 200 μg/ml of H₂O₂ for 30 and 60 minutes

Concentration (μg/mL)	30 minutes	60 minutes
	Mean ± SE	Mean ± SE
0	100.00^A±0.00	100.00^A±0.00
50	86.24^A,B±1.42	84.38^A,B±1.75
100	78.06^B±1.17	73.23^B±3.15
200	76.03^B±0.23	67.89^B±1.66

Notes: Data are presented as % mean ± standard error (n=6). Different superscript letters indicate significant (P<0.001) effects of doses within times of incubation.

Abbreviation: SE, standard error.

Table 5 Cell viability of RAW264.7 cells treated with 1, 10, 100, and 200 μg/ml plant extracts for 24 hours

Extract	Concentration (μg/mL)
	1	10	100	200
	Mean ± SE	Mean ± SE	Mean ± SE	Mean ± SE
AL	98.82^A±1.64	97.78^A±1.73	56.34^B±0.86	44.46^C±1.16
CS	95.97^A±0.00	89.14^A±0.00	56.95^B±0.00	32.97^C±0.00
EA	95.98^A±1.50	93.88^A±1.22	74.14^B±3.19	39.36^C±1.63
ES	89.31^A±0.93	86.59^A±1.44	55.81^B±1.39	34.66^C±1.44
PG	94.21^A±1.44	87.48^A±1.52	55.48^B±1.80	54.44^B±0.90
VM	99.00^A±1.12	95.53^A±2.33	77.95^B±1.08	49.12^C±1.08

Notes: Data are presented as % mean ± standard error (n=6). Different superscript letters indicate significant differences (P<0.001) within treatments at different concentrations (within each row).

Abbreviations: AL, Arctium lappa; CS, Camellia sinensis; EA, Echinacea angustifolia; ES, Eleutherococcus senticosus; PG, Panax ginseng; SE, standard error; VM, Vaccinium myrtillus.